Compositions and methods for production and use of an injectable naturally secreted extracellular matrix

ABSTRACT

The present invention discloses compositions containing natural human extracellular matrices and methods for the use thereof. More particularly, the present invention provides compositions and methods for the repair of skin defects using natural human extracellular matrix by injection.

[0001] This application is a continuation-in-part application of U.S.patent application Ser. No. 08/470,101 filed Jun. 6, 1995, which isincorporated by reference herein in its entirety.

1. INTRODUCTION

[0002] The present invention relates to compositions and methods for thetreatment and repair of soft tissue and skin defects such as wrinklesand scars. More particularly, the invention relates to an injectablecomposition of human extracellular matrix components and methods ofpreparing and using same. The injectable preparation is obtained fromthree-dimensional living stromal tissues that are prepared in vitro.

2. BACKGROUND OF THE INVENTION

[0003] The idea of using an injectable material for soft tissueaugmentation and repair developed soon after the invention of thehypodermic needle. Various products have been injected into the humanbody for correction of soft tissue and skin defects including paraffin,petrolatum, vegetable oils, lanolin, bees wax, and silicone. Injectableliquid silicone has been used extensively, however, due to long termside effects, such as nodules, recurring cellulitis and skin ulcerswhich are now being followed more closely, the use of injectablesilicone is on the decline. Further, in the State of Nevada it is afelony to use injectable silicone in a human. Orange, Skin and AllergyNews (1992) Vol.23, No.6, pg. 1. More recently, bovine collagen hasgained widespread use as an injectable material for soft tissueaugmentation. Collagen is the principal extracellular structural proteinof the animal body. At least fourteen types of mammalian collagen havebeen described. The common characteristic amongst them is a threestranded helix, consisting of three polypeptide chains, calledalpha-chains. All alpha-chains have the same configuration, but differin the composition and sequence of their amino acids. Although thisleads to different types of alpha-chains, however, they all have glycineat every third position in the amino acid sequence. The glycine at everythird position allows for the helical structure of the alpha-chains.Type I collagen is composed of two alpha₁-chains and one alpha₂-chainand is the principal extracellular material of skin, tendon and bone.When clinicians mention “collagen”, they are usually referring to type Icollagen. See Table I, infra, for a detailed listing of collagen typesI-V and in which tissues they are found.

[0004] Collagen has been used as an implant material to replace oraugment hard or soft connective tissue, such as skin, tendon, cartilage,bone and interstitium. Additionally, collagen implants have been usedfor cosmetic purposes for a number of years since collagen can helpcellular ingrowth at the placement site. Early collagen implants wereoften solid collagen masses which were cross-linked with chemicalagents, radiation or other means to improve mechanical properties,decrease immunogenicity and/or increase resistance to resorption. Thecollagen utilized was in a variety of forms, including cross-linked andnon-cross-linked fibrillar collagens, gelatins, and the like andsometimes was combined with various other components, such aslubricants, osteogenic factors and the like, depending on use. A majordisadvantage of solid cross-linked collagen implants is the requirementfor surgical implantation by means of incision. In addition, lack ofdeformability and flexibility are other disadvantages of solid collagenimplants.

[0005] Oliver et al., Clinical Orthopaedics & Related Research (1976)115:291-302; Br. J. Exp. Path. (1980) 61:544-549; and Conn. Tissue Res.(1981) 9:59-62 describe implants made by treating skin with trypsinfollowed by cross-linking with an aldehyde. The resulting solid collagenimplants were reported to maintain their original mass after prolongedimplantation. A main problem with such solid implants is that they mustbe implanted surgically. Other disadvantages are that they are not asdeformable as injectable implants and residual glutaraldehyde may causethe implant to lose its flexibility due to continuing cross-linking insitu.

[0006] Schechter, et al., Br. J. Plas. Surg. (1975) 28:198-202 discloseglutaraldehyde cross-linked skin that was soaked in L-alanine aftercross-linking. The article postulates that the exposure of the skin toL-alanine blocked residual reactive groups of the aldehyde, therebypreventing the release of toxic molecules generated by such groups.

[0007] An alternative to surgically implanted solid collagen material isdisclosed in U.S. Pat. No. 3,949,073. U.S. Pat. No. 3,949,073 describesthe use of atelopeptide solutions of bovine collagen as an injectableimplant material for augmenting soft tissue. According to the patent,the bovine collagen is reconstituted before implantation and forms afibrous mass of tissue when implanted. The patent suggests addingparticles of insoluble bovine collagen microfibrils to control theshrinkage of the fibrous mass formed at the augmentation site. Thecommercial embodiment of the material described in the patent iscomposed of reconstituted atelopeptide bovine collagen in saline thatcontains a small amount of local anesthetic. While effective, theimplant shrinks in volume after implantation due primarily to absorptionof its fluid component by the body. Thus, if volume consistency isessential, an additional injection or injections of supplemental implantmaterial is required. This specific composition has many seriousdrawbacks, e.g., the collagen is from a bovine source, not human, andthe preparation process is not only lengthy and expensive but alsorequires the addition of microfibrils.

[0008] U.S. Pat. No. 4,424,208 describes an injectable dispersion ofcross-linked atelopeptide bovine collagen and reconstituted atelopeptidebovine collagen fibers in an aqueous carrier which exhibited improvedvolume consistency over the material of U.S. Pat. No. 3,949,073.

[0009] U.S. Pat. No. 4,582,640 discloses an improved injectable implantover U.S. Pat. Nos. 3,949,073 and 4,424,208 in which the improvementconsists of improved volume consistency and resistance to physicaldeformation, improved injectability as compared to the dispersion ofU.S. Pat. No. 4,424,208 and that the bovine collagen contains only asingle physical form of collagen as compared to the two physical formsfound in U.S. Pat. No. 4,424,208.

[0010] U.S. Pat. No. 4,803,075 describes bovine collagen compositionsincluding a lubricant material to enhance injectability through narrowdiameter needles for soft tissue repair.

[0011] Despite the advantages and overall usefulness of the injectablecollagen implant-materials disclosed above, problems associated withproducing and injecting the materials have been encountered. Forexample, for soft tissue repair, suspensions of fibrillar collagen haveoften been used by injecting the composition to a treatment site througha fine gauge needle. The use of fibrillar collagen as the primary matrixmaterial in injectable soft and hard tissue implant compositions hasseveral limitations. The preparation of fibrillar collagen suitable forhuman use is relatively time consuming and expensive. In particular, thecomplete removal of contaminating and potentially immunogenic substancesto produce atelocollagen is a relatively complex and expensiveprocedure. Moreover, the persistence, shape retention, cohesiveness,stability, elasticity, toughness and intrudability of the fibrillarcollagen compositions are not optimal.

[0012] In addition to the problems associated with producing andinjecting the collagen implant materials, problems with the actual useof the above mentioned patented injectable implants are also abundant.For instance, since the above patented injectables derive collagen fromxenogeneic sources, usually bovine collagen, the collagen must bemodified to reduce its immunogenicity. Even with modified collagen, theimplant material is still quite immunogenic to which some people areeither already naturally allergic or develop an allergic reaction overtime to the bovine collagen. Due to these allergic reactions theinjectable collagen implants described above cannot be given to manypeople and others are limited to receiving only three injections peryear. Severe allergic reactions include symptoms of rheumatoidarthritis, while less severe reactions include redness and swelling atthe site-of injection which may lead to permanent scarring. Because ofthese severe side effects, the above described collagen injectables areno longer used for lip augmentation. Further, the problems associatedwith injecting xenogeneic collagen seem so intractable that rather thaninjecting collagen, biocompatible ceramic matrices have been injected toachieve similar results as described in U.S. Pat. No. 5,204,382.

[0013] In summary, due to the shortcomings of the above-describedinjectable compositions for the repair of soft tissue defects, such asthe lack of persistence, the need for repeated injections and seriousconcern over adverse reactions, newer injectable materials for softtissue augmentation are needed.

3. SUMMARY OF THE INVENTION

[0014] The present invention relates to injectable materials for softtissue augmentation and methods for use and manufacture of the same,which overcome the shortcomings of bovine injectable collagen and otherinjectable materials, including silicone, of the prior art. Theinjectable materials used in accordance with the present inventioncomprise naturally secreted extracellular matrix preparations as well aspreparations derived from naturally secreted extracellular matrix. Thesepreparations are biocompatible, biodegradable and are capable ofpromoting connective tissue deposition, angiogenesis,reepithilialization and fibroplasia, which is useful in the repair ofskin and other tissue defects. These extracellular matrix preparationsmay be used to repair tissue defects by injection at the site of thedefect.

[0015] The injectable preparations of the present invention have manyadvantages over conventional injectable collagen preparations used forthe repair of skin defects. The extracellular matrix preparations of thepresent invention contain only human proteins, therefore, there is areduced risk of an immune response due to foreign proteins or peptides,especially the type of immune response seen with bovine collagen foundin conventional injectable collagen preparations. Additionally, theinjected preparations of the present invention should persist longer andeven if multiple injections are required, the injections should not besubject to the “no more than three injections per year” rule of bovinecollagen-based preparations due to the lack of immunogenicity. Anotheradvantage provided by the present invention is that the preparations ofnative extracellular matrix contain a mixture of extracellular matrixproteins which closely mimics the compositions of physiologically normalconditions, for example, in an extracellular matrix derived from dermalcells, type I and III collagens, hyaluronic acid as well as variousglycosaminoglycans and natural growth factors are present. Many of theseextracellular matrix proteins and growth factors have been studiedextensively and have been shown to be critical for wound healing andtissue restoration.

[0016] In another aspect of the invention, the preparations can be usedin highly improved systems for in vitro tissue culture. In thisembodiment, naturally secreted extracellular matrix coatedthree-dimensional frameworks can be used to culture cells which requireattachment to a support in order to grow but do not attach toconventional tissue culture vessels. In addition to culturing cells on acoated framework, the extracellular matrix secreted by the cells ontothe framework can be collected and used to coat vessels for use intissue culture. The extracellular matrix, acting as a base substrate,may allow cells normally unable-to attach to conventional tissue culturedish base substrates to attach and subsequently grow.

[0017] Yet another embodiment of the present invention is directed to anovel method for determining the ability for cellular taxis of aparticular cell. The method involves inoculating one end of a nativeextracellular matrix coated three-dimensional framework with the celltype in question and over time measure the distance traversed across theframework by the cell. Because the extracellular matrix is secretednaturally by the cells onto the framework, it is an excellent in vitroequivalent of extracellular matrix found in the body. Such an assay, forexample, may inform whether isolated tumor cells are metastatic orwhether certain immune cells can migrate across or even chemotact acrossthe framework, thus, indicating that the cell has such cellular taxisability.

3.1. DEFINITIONS AND ABBREVIATIONS

[0018] The following terms used herein shall have the meaningsindicated:

[0019] Adherent Layer:

[0020] cells attached directly to the three-dimensional framework orconnected indirectly by attachment to cells that are themselves attacheddirectly to the matrix.

[0021] Pharmaceutically Accentable Carrier:

[0022] an aqueous medium at physiological isotonicity and pH and maycontain other elements such as local anesthetics and/or fluidlubricants.

[0023] Stromal Cells:

[0024] fibroblasts with or without other cells and/or elements found inloose connective tissue, including but not limited to, endothelialcells, pericytes, macrophages, monocytes, plasma cells, mast cells,adipocytes, chondrocytes, etc.

[0025] Three-Dimensional Framework:

[0026] a three dimensional support composed of any material and/or shapethat (a) allows cells to attach to it (or can be modified to allow cellsto attach to it); and (b) allows cells to grow in more than one layer.This support is inoculated with stromal cells to form the living stromalmatrix.

[0027] Living Stromal Tissue:

[0028] a three dimensional framework which has been inoculated withstromal cells. Whether confluent or subconfluent, stromal cellsaccording to the invention continue to grow and divide. The livingstromal tissue prepared in vitro is the source of the extracellularmatrix proteins used in the injectable formulations of the invention.

[0029] The following abbreviations shall have the meanings indicated:

[0030] EDTA ethylene diamine tetraacetic acid

[0031] FBS fetal bovine serum

[0032] HBSS Hank's balanced salt solution

[0033] HS horse serum

[0034] MEM minimal essential medium

[0035] PBS phosphate buffered saline

[0036] RPMI 1640 Roswell Park Memorial Institute Medium No. 1640 (GIBCO,Inc., Grand Island, N.Y.)

[0037] SEM scanning electron microscopy

[0038] The present invention may be more fully understood by referenceto the following detailed description, examples of specific embodimentsand appended figures which are offered for purposes of illustration onlyand not by way of limitation.

4. BRIEF DESCRIPTION OF THE FIGURES

[0039]FIG. 1. FIG. 1 is a scanning electron micrograph depictingfibroblast attachment to the three-dimensional matrix and extension ofcellular processes across the mesh opening. Fibroblasts are activelysecreting matrix proteins and are at the appropriate stage ofsubconfluency which should be obtained prior to inoculation withtissue-specific cells.

[0040] FIGS. 2A-D. FIGS. 2A-D are transmission electron micrographs ofcollagen isolated from extracellular matrix prepared from dermal tissuegrown in vitro (FIG. 2A-B) or from a normal adult human dermal sample(FIGS. 2C-D).

5. DETAILED DESCRIPTION OF THE INVENTION

[0041] One embodiment of the present invention involves the preparationand use of an injectable extracellular matrix composition for thetreatment of skin defects. The extracellular matrix proteins are derivedfrom a living stromal tissue prepared in vitro by growing stromal cellson a three-dimensional framework resulting in a multi-layer cell culturesystem. In conventional tissue culture systems, the cells were grown ina monolayer. Cells grown on a three-dimensional framework support, inaccordance with the present invention, grow in multiple layers, forminga cellular matrix. This matrix system approaches physiologic conditionsfound in vivo to a greater degree than previously described monolayertissue culture systems. The three-dimensional cell culture system isapplicable to the proliferation of different types of stromal cells andformation of a number of different stromal tissues, including but notlimited to dermis, bone marrow stroma, glial tissue, cartilage, to namebut a few.

[0042] In accordance with the present invention, the pre-establishedliving stromal tissue comprises stromal cells grown on athree-dimensional framework or network. The stromal cells can comprisefibroblasts with or without additional cells and/or elements describedmore fully herein. The fibroblasts and other cells and/or elements thatcomprise the stroma can be fetal or adult in origin, and can be derivedfrom convenient sources such as skin, liver, pancreas, etc. Such tissuesand/or organs can be obtained by appropriate biopsy or upon autopsy. Infact, cadaver organs may be used to provide a generous supply of stromalcells and elements.

[0043] Once inoculated onto the three-dimensional framework, the stromalcells will proliferate on the framework, and elaborate growth factors,regulatory factors and extracellular matrix proteins that are depositedon the support. The living stromal tissue will sustain activeproliferation of the culture for long periods of time. Growth andregulatory factors can be added to the culture, but are not necessarysince they are elaborated by the stromal support matrix.

[0044] The naturally secreted extracellular matrix is collected from thethree-dimensional framework and is processed further with apharmaceutically acceptable aqueous carrier and placed in a syringe forprecise placement of the biomaterial into tissues, such as the facialdermis.

[0045] The present invention is based, in part, on the discovery thatduring the growth of human stromal cells on a biodegradable ornon-biodegradable three-dimensional support framework, the cellssynthesize and deposit on the three-dimensional support framework ahuman extracellular matrix as produced in normal human tissue. Theextracellular matrix is secreted locally by cells and not only bindscells and tissues together but also influences the development andbehavior of the cells it contacts. The extracellular matrix containsvarious fiber-forming proteins interwoven in a hydrated gel composed ofa network of glycosaminoglycan chains. The glycosaminoglycans are aheterogeneous group of long, negatively charged polysaccharide chains,which (except for hyaluronic acid) are covalently linked to protein toform proteoglycan molecules.

[0046] The fiber-forming proteins are of two functional types: mainlystructural (collagens and elastin) and mainly adhesive (such asfibronectin and laminin). The fibrillar collagens (types I, II, and III)are rope-like, triple-stranded helical molecules that aggregate intolong cable-like fibrils in the extracellular space; these in turn canassemble into a variety of highly ordered arrays. Type IV collagenmolecules assemble into a sheetlike meshwork that forms the core of allbasal laminae. Elastin molecules form an extensive cross-linked networkof fibers and sheets that can stretch and recoil, imparting elasticityto the matrix. Fibronectin and laminin are examples of large adhesiveglycoproteins in the matrix; fibronectin is widely distributed inconnective tissues, whereas laminin is found mainly in basal laminae. Bymeans of their multiple binding domains, such proteins help cells adhereto and become organized by the extracellular matrix.

[0047] As an example, a naturally secreted human dermal extracellularmatrix contains type I and type III collagens, fibronectin, tenascin,glycosaminoglycans, acidic and basic FGF, TGF-α and TGF-β, KGF, decorinand various other secreted human dermal matrix proteins. As naturallysecreted products, the various extracellular matrix proteins areproduced in the quantities and ratios similar to that existing in vivo.Moreover, growth of the stromal cells in three dimensions will sustainactive proliferation of cells in culture for much longer time periodsthan will monolayer systems. Further, the three-dimensional systemsupports the maturation, differentiation, and segregation of cells inculture in vitro to form components of adult tissues analogous tocounterparts found in vivo. Thus, the extracellular matrix created bythe cells in culture is more analogous to native tissues.

[0048] Although the applicants are under no duty or obligation toexplain the mechanism by which the invention works, a number of factorsinherent in the three-dimensional culture system may contribute to thesefeatures of the three dimensional culture system:

[0049] (a) The three-dimensional framework provides a greater surfacearea for protein attachment, and consequently, for the adherence ofstromal cells.

[0050] (b) Because of the three-dimensionality of the framework, stromalcells continue to actively grow in contrast to cells in monolayercultures, which grow to confluence, exhibit contact inhibition, andcease to grow and divide. The elaboration of growth and regulatoryfactors by replicating stromal cells may be partially responsible forstimulating proliferation and regulating differentiation of cells inculture.

[0051] (c) The three-dimensional framework allows for a spatialdistribution of cellular elements which is more analogous to that foundin the counterpart tissue in vivo.

[0052] (d) The increase in potential volume for cell growth in thethree-dimensional system may allow the establishment of localizedmicroenvironments analogous to native counterparts found in vivo.

[0053] (e) The three-dimensional matrix maximizes cell-cell interactionsby allowing greater potential for movement of migratory cells, such asmacrophages, monocytes and possibly lymphocytes in the adherent layer.

[0054] (f) It has been recognized that maintenance of a differentiatedcellular phenotype requires not only growth/differentiation factors butalso the appropriate cellular interactions. The present inventioneffectively recreates the stromal tissue microenvironment.

[0055] The three-dimensional stromal support, the culture system itself,and its maintenance, as well as various uses of the three-dimensionalcultures and of the naturally secreted extracellular matrix aredescribed in greater detail in the subsections below. Solely for ease ofexplanation, the detailed description of the invention is divided intothe three sections, (i) growth of the three-dimensional stromal cellculture, (ii) isolation of the naturally secreted human extracellularmatrix, and (iii) formulation of the isolated extracellular matrix intopreparations for injection at the site of soft tissue defects.

[0056] 5.1. Preparing the Living Stromal Tissue In Vitro

[0057] The three-dimensional support used to culture stromal tissue maybe of any material and/or shape that:

[0058] (a) allows cells to attach to it (or can be modified to allowcells to attach to it); and

[0059] (b) allows cells to grow in more than one layer.

[0060] A number of different materials may be used to form theframework, such as non-biodegradable or biodegradable materials. Forexample, non-biodegradable materials include but are not limited to:nylon (polyamides), dacron (polyesters), polystyrene, polypropylene,polyacrylates, polyvinyl compounds (e.g., polyvinylchloride),polycarbonate (PVC), polytetrafluorethylene (PTFE; teflon), thermanox(TPX), etc. Additionally, biodegradable material may also be utilized,including but not limited to: nitrocellulose, cotton, polyglycolic acid(PGA), cat gut sutures, cellulose, gelatin, dextran, collagen, chitosan,hyaluronic acid, etc. Any of these materials, bio- or non-biodegradable,can be woven into a mesh to form a three-dimensional framework.Alternatively, the materials can be used to form other types ofthree-dimensional frameworks, for example, sponges, such as collagensponges.

[0061] Certain materials, such as nylon, polystyrene, etc., are poorsubstrates for cellular attachment. When these materials are used as thethree-dimensional support framework, it is advisable to pre-treat theframework prior to inoculation of stromal cells in order to enhance theattachment of stromal cells to the framework. For example, prior toinoculation with stromal cells, nylon frameworks can be treated with 0.1M acetic acid, and incubated in polylysine, FBS, and/or collagen to coatthe nylon. Polystyrene can be similarly treated using sulfuric acid. Aconvenient nylon mesh which can be used in accordance with the inventionis Nitex, a nylon filtration mesh having an average pore size of 210 μmand an average nylon fiber diameter of 90 μm (#3-210/36, Tetko, Inc.,N.Y.).

[0062] Stromal cells comprising fibroblasts derived from adult or fetaltissue, with or without other cells and elements described below, areinoculated onto the framework. These fibroblasts may be derived fromorgans, such as skin, liver, pancreas, etc. which can be obtained bybiopsy, where appropriate, or upon autopsy. In fact, fibroblasts can beobtained in quantity rather conveniently from any appropriate cadaverorgan. In a preferred embodiment, fetal fibroblasts can be obtained inhigh quantity from foreskin.

[0063] Fibroblasts may be readily isolated by disaggregating anappropriate organ or tissue which is to serve as the source of thefibroblasts. This can be readily accomplished using techniques known tothose skilled in the art. For example, the tissue or organ can bedisaggregated mechanically and/or treated with digestive enzymes and/orchelating agents that-weaken the connections between neighboring cellsmaking it possible to disperse the tissue into a suspension ofindividual cells without appreciable cell breakage. Enzymaticdissociation can be accomplished by mincing the tissue and treating theminced tissue with any of a number of digestive enzymes either alone orin combination. These include but are not limited to trypsin,chymotrypsin, collagenase, elastase, hyaluronidase, DNase, pronase,and/or dispase etc. Mechanical disruption can also be accomplished by anumber of methods including, but not limited to the use of grinders,blenders, sieves, homogenizers, pressure cells, or insonators to namebut a few. For a review of tissue disaggregation techniques, seeFreshney, Culture of Animal Cells. A Manual of Basic Technique, 2d Ed.,A. R. Liss, Inc., New York, 1987, Ch. 9, pp. 107-126.

[0064] Once the tissue has been reduced to a suspension of individualcells, the suspension can be fractionated into subpopulations from whichthe fibroblasts and/or other stromal cells and/or elements can beobtained. This also may be accomplished using standard techniques forcell separation including, but not limited to, cloning and selection ofspecific cell types, selective destruction of unwanted cells (negativeselection), separation based upon differential cell agglutinability inthe mixed population, freeze-thaw procedures, differential adherenceproperties of the cells in the mixed population, filtration,conventional and zonal centrifugation, centrifugal elutriation(counter-streaming centrifugation), unit gravity separation,countercurrent distribution, electrophoresis and fluorescence-activatedcell sorting. For a review of clonal selection and cell separationtechniques, see Preshney, Culture of Animal Cells. A Manual of BasicTechniques, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 11 and 12, pp.137-168.

[0065] The isolation of fibroblasts, for example, can be carried out asfollows: fresh tissue samples are thoroughly washed and minced in Hanks'balanced salt solution (HBSS) in order to remove serum. The mincedtissue is incubated from 1-12 hours in a freshly prepared solution of adissociating enzyme such as trypsin. After such incubation, thedissociated cells are suspended, pelleted by centrifugation and platedonto culture dishes. All fibroblasts will attach before other cells,therefore, appropriate stromal cells can be selectively isolated andgrown. The isolated fibroblasts can then be grown to confluency, liftedfrom the confluent culture and inoculated onto the three-dimensionalframework, see Naughton et al., 1987, J. Med. 18(3&4):219-250.Inoculation of the three-dimensional framework with a high concentrationof stromal cells, e.g., approximately 10⁶ to 5×10⁷ cells/ml, will resultin the establishment of the three-dimensional stromal support in shorterperiods of time.

[0066] In addition to fibroblasts, other cells can be added to form thethree-dimensional stromal cell culture-producing extracellular matrix.For example, other cells found in loose connective tissue may beinoculated onto the three-dimensional support framework along withfibroblasts. Such cells include, but are not limited to, endothelialcells, pericytes, macrophages, monocytes, plasma cells, mast cells,adipocytes, chondrocytes, etc. These stromal cells can be readilyderived from appropriate organs such as skin, liver, etc., using methodsknown, such as those discussed above.

[0067] In one embodiment of the present invention, stromal cells whichare specialized for the particular tissue to be cultured can be added tothe fibroblast stroma for the production of a tissue type specificextracellular matrix. For example, dermal fibroblasts can be used toform the three-dimensional subconfluent stroma for the production ofskin-specific extracellular matrix in vitro. Alternatively, stromalcells of hematopoietic tissue including, but not limited to, fibroblastendothelial cells, macrophages/monocytes, adipocytes and reticularcells, can be used to form the three-dimensional subconfluent stroma forthe production of a bone marrow-specific extracellular matrix in vitro,see infra. Hematopoietic stromal cells can be readily obtained from the“buffy coat” formed in bone marrow suspensions by centrifugation at lowforces, e.g., 3000×g. Stromal cells of liver may include fibroblasts,Kupffer cells, and vascular and bile duct endothelial cells. Similarly,glial cells can be used as the stroma to support the proliferation ofneurological cells and tissues. Glial cells for this purpose can beobtained by trypsinization or collagenase digestion of embryonic oradult brain. Ponten and Westermark, 1980, In Federof, S. Hertz, L., eds,“Advances in Cellular Neurobiology,” Vol.1, New York, Academic Press,pp.209-227.

[0068] For certain uses in vivo it is preferable to obtain the stromalcells from the patient's own tissues. The growth of cells in thepresence of the three-dimensional stromal support framework can befurther enhanced by adding to the framework, or coating the frameworksupport with proteins, e.g., collagens, elastic fibers, reticularfibers, glycoproteins; glycosaminoglycans, e.g., heparin sulfate,chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratansulfate, etc.; a cellular matrix, and/or other materials.

[0069] After inoculation of the stromal cells, the three-dimensionalframework is incubated in an appropriate nutrient medium underphysiologic conditions favorable for cell growth, i.e., promotingmitosis, i.e., cell division. Many commercially available media such asRPMI 1640, Fisher's, Iscove's, McCoy's, and the like may be suitable foruse. It is important that the three-dimensional stromal culture besuspended or floated in the medium during the incubation period in orderto maximize proliferative activity. In addition, the culture should be“fed” periodically to remove the spent media, depopulate released cells,and to add fresh media.

[0070] During the incubation period, the stromal cells will growlinearly along and envelop the three-dimensional framework beforebeginning to grow into the openings of the framework. The cells aregrown to an appropriate degree to allow for adequate deposition ofextracellular matrix proteins.

[0071] The openings of the framework should be of an appropriate size toallow the stromal cells to stretch across the openings. Maintainingactively growing stromal cells which stretch across the frameworkenhances the production of growth factors which are elaborated by thestromal cells, and hence, will support long term cultures. For example,if the openings are too small, the stromal cells may rapidly achieveconfluence but be unable to easily exit from the mesh. Trapped cells canexhibit contact inhibition and cease production of the appropriatefactors necessary to support proliferation and maintain long termcultures. If the openings are too large, the stromal cells are unable tostretch across the opening. This will also decrease stromal cellproduction of the appropriate factors necessary to support proliferationand maintain long term cultures. When using a mesh type of matrix, asexemplified herein, we have found that openings ranging from about 150μm to about 220 μm will work satisfactorily. However, depending upon thethree-dimensional structure and intricacy of the framework, other sizesmay work equally well. In fact, any shape or structure that allows thestromal cells to stretch and continue to replicate and grow for lengthytime periods will work in accordance with the present invention.

[0072] Different proportions of the various types of collagen depositedon the framework can be achieved by inoculating the framework withdifferent tissue-specific cells. For example, for hematopoietic cells,the matrix should preferably contain collagen types III, IV and I in anapproximate ratio of 6:3:1 in the initial matrix. For skin, collagentypes I and III are preferably deposited in the initial matrix. Theproportions of collagen types deposited can be manipulated or enhancedby selecting fibroblasts which elaborate the appropriate extracellularmatrix proteins. This can be accomplished using monoclonal antibodies ofan appropriate isotype or subclass which are capable of activatingcomplement, and which define particular collagen types. These antibodiesin combination with complement can be used to negatively select thefibroblasts which express the desired collagen type. Alternatively, thestroma used to inoculate the framework can be a mixture of cells whichsynthesize the appropriate collagen types desired. The distribution andorigins of the five types of collagen is shown in Table I. TABLE IDISTRIBUTIONS AND ORIGINS OF THE FIVE TYPES OF COLLAGEN CollagenPrincipal Tissue Type Distribution Cells of Origin I Loose and denseordinary Fibroblasts and connective tissue; collagen reticular cells;fibers smooth muscle cells Fibrocartilage Bone Osteoblast DentinOdontoblasts II Hyaline and elastic Chondrocytes cartilage Retinal CellsVitreous body of eye III Loose connective tissue; Fibroblasts andreticular fibers reticular cells Papillary layer of dermis Smooth musclecells; Blood vessels endothelial cells IV Basement membranes Epithelialand endothelial cells Lens capsule of eye Lens fibers V Fetal membranes;placenta Fibroblasts Basement membranes Bone Smooth muscle Smooth musclecells

[0073] Thus, depending upon the collagen types desired, the appropriatestromal cell(s) can be selected to inoculate the three-dimensionalframework.

[0074] The three-dimensional extracellular matrix producing culture ofthe present invention affords a vehicle for introducing gene products invivo. In certain situations, it may be desirable to prepare anextracellular matrix containing a foreign gene product, growth factor,regulatory factor, etc. In such cases, the cells may be geneticallyengineered to express the gene product, or altered forms of the geneproduct that are immobilized in the extracellular matrix laid down bythe stromal cells. For example, using recombinant DNA techniques, a geneof interest can be placed under the control of an inducible promoter.The recombinant DNA construct containing the gene can be used totransform or transfect a host cell which is cloned and then clonallyexpanded in the three-dimensional culture system. The use of thethree-dimensional culture in this regard has a number of advantages.First, since the culture comprises eukaryotic cells, the gene productwill be properly expressed and processed in culture to form an activeproduct. Second, the number of transfected cells can be substantiallyenhanced to be of clinical value, relevance, and utility. Thethree-dimensional cultures of the present invention allow for expansionof the number of transfected cells and amplification (via cell division)of transfected cells.

[0075] Preferably, the expression control elements used should allow forthe regulated expression of the gene so that the product can be oversynthesized in culture. The transcriptional promoter chosen, generally,and promoter elements specifically, depends, in part, upon the type oftissue and cells cultured. Cells and tissues which are capable ofsecreting proteins (e.g., those characterized by abundant roughendoplasmic reticulum and golgi complex) are preferable.

[0076] During incubation of the three-dimensional culture, proliferatingcells are released from the framework. These released cells can stick tothe walls of the culture vessel where they can continue to proliferateand form a confluent monolayer. This should be prevented or minimized,for example, by removal of the released cells during feeding, or bytransferring the three-dimensional framework to a new culture vessel.The presence of a confluent monolayer in the vessel will “shut down” thegrowth of cells in the three-dimensional framework and/or culture.Removal of the confluent monolayer or transfer of the stromal culture tofresh media in a new vessel will restore proliferative activity of thethree-dimensional culture system. Such removal or transfers should bedone in any culture vessel which has a stromal monolayer exceeding 25%confluency. Alternatively, the culture system can be agitated to preventthe released cells from sticking, or instead of periodically feeding thecultures, the culture system could be set up so that fresh mediacontinuously flows through the system. The flow rate can be adjusted toboth maximize proliferation within the three-dimensional culture, and towash out and remove cells released from the matrix, so that they willnot stick to the walls of the vessel and grow to confluence. In anycase, the released stromal cells can be collected and crypreserved forfuture use.

[0077] Once inoculated onto the three-dimensional framework, adherenceof the fibroblasts is seen quickly (e.g., within hours) and thefibroblasts begin to stretch across the framework openings within days.These fibroblasts are metabolically active, secrete extracellular matrixand rapidly form a dermal equivalent consisting of active fibroblastsand collagen.

[0078]FIG. 1 illustrates the ability of the fibroblasts to arrangethemselves into parallel layers between the naturally-secreted collagenbundles. These fibroblasts exhibit a rapid rate of cell division andprotein secretion.

[0079] 5.2. Removal of the Extracellular Matrix from the Framework

[0080] After the cells have been inoculated onto the framework andcultured under conditions favoring cellular growth, such that a desiredamount of extracellular matrix is secreted on to the three-dimensionalframework, the cells are killed and the naturally secreted extracellularmatrix is processed further.

[0081] This involves first killing the cells and removing the killedcells and any cellular debris from the three-dimensional framework. Thisprocess is carried out in a number of different ways. For example, thecells can be killed by flash-freezing the living stromal tissue preparedin vitro in liquid nitrogen without a cryopreservative. Another way tokill the cells is to irrigate the inoculated three-dimensional frameworkwith sterile water, such that the cells burst in response to osmoticpressure. Once the cells have been killed, one can, for example, disruptthe cellular membranes and remove the cellular debris by a milddetergent rinse, such as EDTA, CHAPS or a zwitterionic detergent,followed by treatment with a cryoprotectant such as DMSO, propyleneglycol, butanediol, raffinose, polyvinyl pyrrolidone, dextran or sucroseand vitrified in liquid nitrogen. Alternatively, the framework can besubjected to enzymatic digestion and/or extracting with reagents thatbreak down the cellular membranes and allow removal of cell contents.Examples of detergents include non-ionic detergents (for example, TRITONX-100, octylphenoxy polyethoxyethanol, (Rohm and Haas); BRIJ-35, apolyethoxyethanol lauryl ether (Atlas Chemical Co.), TWEEN 20, apolyethoxyethanol sorbitan monolaureate (Rohm and Haas), LUBROL-PX, orpolyethylene lauryl ether (Rohm and Haas)); and ionic detergents (forexample, sodium dodecyl sulphate, sulfated higher aliphatic alcohol,sulfonated alkane and sulfonated alkylarene containing 7 to 22 carbonatoms in a branched or unbranched chain). Enzymes can be used also andcan include nucleases (for example, deoxyribonuclease and ribonuclease),phospholipases and lipases. An advantage to using a mild detergent rinseis that it will solubilize membrane-bound proteins, which are oftenhighly antigenic.

[0082] Once the cells have been killed and the cellular debris has beenremoved, the collection of the naturally secreted human extracellularmatrix can be accomplished in a variety of ways which depends on whetherthe three-dimensional framework is composed of material that isbiodegradable or non-biodegradable. For example, if the framework iscomposed of non-biodegradable material, one can remove the extracellularmatrix from a non-biodegradable support by subjecting thethree-dimensional framework to sonication and/or to high pressure waterjets and/or to mechanical scraping and/or to a mild treatment withdetergents and/or enzymes to remove the attached extracellular matrixfrom the framework.

[0083] If the extracellular matrix is deposited on a biodegradablethree-dimensional framework, after killing and removing the cells andcellular debris, the extracellular matrix can be recovered, for example,by simply allowing the framework to degrade in solution, i.e., allow theframework to dissolve, thus freeing the extracellular matrix.Additionally, if the biodegradable support is composed of a materialwhich can be injected, like the extracellular matrix itself, one canprocess the entire extracellular matrix coated framework into syringesfor injection. Further, if the extracellular matrix is deposited on abiodegradable support, the matrix can be removed by the same methods asif the matrix had been deposited on a non-biodegradable support, i.e.,by subjecting the three-dimensional framework to sonication and/or tohigh pressure water jets and/or to mechanical scraping and/or to a mildtreatment with detergents and/or enzymes to remove the attachedextracellular matrix from the framework. None of the removal processesare designed to damage and/or denature the naturally secreted humanextracellular matrix produced by the cells.

[0084] 5.3. Formulation and Use of Injectable Preparations

[0085] Once the naturally secreted extracellular matrix has beencollected, it is processed further. The extracellular matrix can behomogenized to fine particles, such that it can pass through a surgicalneedle. Homogenization is well known in the art, for example, bysonication. Further, the extracellular matrix can be cross-linked bygamma irradiation without the use of chemical cross-linking agents, suchas glutaraldehyde, which are toxic. The gamma irradiation should be aminimum of 20 M rads to sterilize the material since all bacteria,fungi, and viruses are destroyed at 0.2 M rads. Preferably, theextracellular matrix can be irradiated from 0.25 to 2 M rads tosterilize and cross-link the extracellular matrix.

[0086] Further, the amounts and/or ratios of the collagens and otherproteins may be adjusted by mixing extracellular matrices secreted byother cell types prior to placing the material in a syringe. Forexample, biologically active substances, such as proteins and drugs, canbe incorporated in the compositions of the present invention for releaseor controlled release of these active substances after injection of thecomposition. Exemplary biologically active substances can include tissuegrowth factors, such as TGF-β, and the like which promote healing andtissue repair at the site of the injection.

[0087] Final formulation of the aqueous suspension of naturally secretedhuman extracellular matrix will typically involve adjusting the ionicstrength of the suspension to isotonicity (i.e., about 0.1 to 0.2) andto physiological pH (i.e., about pH 6.8 to 7.5) and adding a localanesthetic, such as lidocaine, (usually at a concentration of about 0.3%by weight) to reduce local pain upon injection. The final formulationwill also typically contain a fluid lubricant, such as maltose, whichmust be tolerated by the body. Exemplary lubricant components includeglycerol, glycogen, maltose and the like organic polymer base materials,such as polyethylene glycol and hyaluronic acid as well as non-fibrillarcollagen, preferably succinylated collagen, can also act as lubricants.Such lubricants are generally used to improve the injectability,intrudability and dispersion of the injected biomaterial at the site ofinjection and to decrease the amount of spiking by modifying theviscosity of the compositions. This final formulation is by definitionthe processed extracellular matrix in a pharmaceutically acceptablecarrier.

[0088] The matrix is subsequently placed in a syringe or other injectionapparatus for precise placement of the matrix at the site of the tissuedefect. In the case of formulations for dermal augmentation, the term“injectable” means the formulation can be dispensed from syringes havinga gauge as low as 25 under normal conditions under normal pressurewithout substantial spiking. Spiking can cause the composition to oozefrom the syringe rather than be injected into the tissue. For thisprecise placement, needles as fine as 27 gauge (200μ I.D.) or even 30gauge (150μ I.D.) are desirable. The maximum particle size that can beextruded through such needles will be a complex function of at least thefollowing: particle maximum dimension, particle aspect ratio(length:width), particle rigidity, surface roughness of particles andrelated factors affecting particle:particle adhesion, the viscoelasticproperties of the suspending fluid, and the rate of flow through theneedle. Rigid spherical beads suspended in a Newtonian fluid representthe simplest case, while fibrous or branched particles in a viscoelasticfluid are likely to be more complex.

[0089] The above described steps in the process for preparing injectablenaturally secreted human extracellular matrix are preferably carried outunder sterile conditions using sterile materials. The processedextracellular matrix in a pharmaceutically acceptable carrier can beinjected intradermally or subcutaneously to augment soft tissue, torepair or correct congenital anomalies, acquired defects or cosmeticdefects. Examples of such conditions are congenital anomalies ashemifacial microsomia, malar and zygomatic hypoplasia, unilateralmammary hypoplasia, pectus excavatum, pectoralis agenesis (Poland'sanomaly) and velopharyngeal incompetence secondary to cleft palaterepair or submucous cleft palate (as a retropharyngeal implant);acquired defects (post-traumatic, post-surgical, post-infectious) suchas depressed scars, subcutaneous atrophy (e.g., secondary to discoidlupis erythematosus), keratotic lesions, enophthalmos in the unucleatedeye (also superior sulcus syndrome), acne pitting of the face, linearscleroderma with subcutaneous atrophy, saddle-nose deformity, Romberg'sdisease and unilateral vocal cord paralysis; and cosmetic defects suchas glabellar frown lines, deep nasolabial creases, circum-oralgeographical wrinkles, sunken cheeks and mammary hypoplasia. Thecompositions of the present invention can also be injected into internaltissues, such as the tissues defining body sphincters to augment suchtissues.

[0090] Various sample embodiments of the invention are described in thesections below. For purposes of description only, and not by way oflimitation, the three-dimensional culture system of the invention isdescribed based upon the type of tissue and cells used in varioussystems. These descriptions specifically include but are not limited tobone marrow, skin, epithelial cells, and cartilage but it is expresslyunderstood that the three-dimensional culture system can be used withother types of cells and tissues. The invention is also illustrated byway of examples, which demonstrate characteristic data generated foreach system described.

EXAMPLES

[0091] 6. Example: Three-Dimensional Skin Stromal Culture System

[0092] The subsections below describe the three-dimensional culturesystem of the invention for culturing different stromal cells in vitro.Briefly, cultures of fibroblasts were established on nylon mesh whichhad been previously sterilized. Within 6-9 days of incubation, adherentfibroblasts began to grow into the meshwork openings and depositedparallel bundles of collagen. Indirect immunofluorescence usingmonoclonal antibodies showed predominantly type I collagen with sometype III as well.

[0093] 6.1. Establishment of the Three-Dimensional Stroma of SkinFibroblasts

[0094] Skin fibroblasts were isolated by mincing dermal tissue,trypsinization for-2 hours, and separation of cells into a suspension byphysical means. Fibroblasts were grown to confluency in 25 cm² Falcontissue culture dishes and fed with RPMI 1640 (Sigma, Mo.) supplementedwith 10% fetal bovine serum (PBS), fungizone, gentamicin, andpenicillin/streptomycin. Fibroblasts were lifted by mild trypsinizationand cells were plated onto nylon filtration mesh, the fibers of whichare approximately 90 μm in diameter and are assembled into a squareweave with a mesh opening of 210 μm (Tetko, Inc., NY). The mesh waspretreated with a mild acid wash and incubated in polylysine and FBS.Adherence of the fibroblasts was seen within 3 hours, and fibroblastsbegan to stretch across the mesh openings within 5-7 days of initialinoculation. These fibroblasts were metabolically active, secreted anextracellular matrix, and rapidly formed a dermal equivalent consistingof active fibroblasts and collagen. FIG. 1 is a scanning electronmicrograph depicting fibroblast attachment and extension of cellularprocesses across the mesh opening.

[0095] 6.2 Establishment of the Three-Dimensional Bone Marrow StromalCultures

[0096] Bone marrow was aspirated from multiple sites on the posterioriliac crest of hematologically normal adult volunteers after informedconsent was obtained. Specimens were collected into heparinized tubesand suspended in 8 ml of RPMI 1640 medium which was conditioned with 10%FBS and 5-10% HS and supplemented with hydrocortisone, fungizone, andstreptomycin. The cell clumps were disaggregated and divided intoaliquots of 5×10⁶ nucleated cells.

[0097] Nylon filtration screen (#3-210/36, Tetko Inc., NY) was used as athree-dimensional framework to support all stromal cell culturesdescribed in the examples below. The screen consisted of fibers, whichwere 90 μm in diameter, assembled into a square weave pattern with sieveopenings of 210 μm. Stromal cells were inoculated using the protocolsdescribed in Section 6.1. Adherence and subsequent growth of the stromalelements was monitored using inverted phase contrast microscopy andscanning electron microscopy (SEM).

[0098] 6.3 Preparation of the Three-Dimensional Oral Mucosal EpithelialStromal Matrix

[0099] Samples of oral mucosal tissue were obtained from orthodonticsurgical specimens. Tissue was washed three times with fresh MEMcontaining antibiotics (2 ml of antibiotic antimycotic solution fromGIBCO, Cat. #600-5240 AG; and 0.01 ml of gentamicin solution from GIBCOCat. #600-5710 AD per 100 cc MEM), cut into small pieces, then washedwith 0.02% EDTA (w/v). 0.25% trypsin (in PBS without Ca⁺⁺ or Mg⁺⁺) wasadded; after a few seconds, the tissue pieces were removed and placed infresh trypsin (in PBS without Ca⁺⁺ or Mg⁺⁺) and refrigerated at 4° C.overnight. Tissues were removed and placed in fresh trypsin solution,and gently agitated until cell appeared to form a single-cellsuspension. The single-cell suspension-was then diluted in MEMcontaining 10% heat inactivated fetal bovine serum and centrifuged at1400×g for 7 minutes. The supernatant was decanted and the pelletcontaining mucosal epithelial cells was placed into seeding medium.Medium consisted of DMEM with 2% Ultrosen G, 1×L-glutamine,1×non-essential amino acids, penicillin and streptomycin. The cells wereseeded onto a three-dimensional framework. The three-dimensional stromalculture was generated using oral fibroblasts and 8 mm×45 mm pieces ofnylon filtration screen (#3-210/36, Tetko Inc., NY). The mesh was soakedin 0.1 M acetic acid for 30 minutes and treated with 10 mM polylysinesuspension for 1 hour. The meshes were placed in a sterile petri dishand inoculated with 1×10⁶ oral fibroblasts collected as described abovein DMEM complete medium. After 1-2 hours of incubation at 5% CO₂ themeshes were placed in a Corning 25 cm² tissue culture flask, floatedwith an additional 5 ml of medium, and allowed to reach subconfluence,being fed at 3 day intervals. Cultures were maintained in DMEM completemedium at 37° C. and 5% CO₂ in a humidified atmosphere and were fed withfresh medium every 3 days.

[0100] 6.4 Establishment of the Three Dimensional Small VesselEndothelial Stromal Cell Culture

[0101] Small vessel endothelial cells isolated from the brain accordingto the method of Larson et al., 1987, Microvasc. Res. 34:184were-cultured in vitro using T-75 tissue culture flasks. The cells weremaintained in Dulbecco's Modified Eagle Medium/Hams-F-12 mediumcombination (the solution is available as a 1:1 mixture). The medium wassupplemented with 20% heat-inactivated fetal calf serum (FCS),glutamine, and antibiotics. The cells were seeded at a concentration of1×10⁶ cells per flask, and reached a confluent state within one week.The cells were passaged once a week, and, in addition, were fed once aweek with DMEM/Hams-F-12 containing FCS, glutamine, and antibiotics asdescribed. To passage the cells, flasks were rinsed twice with 5 ml ofPBS (without Ca⁺⁺ or Mg⁺⁺) and trypsinized with 3 ml of 0.05% Trypsinand 0.53 mM EDTA. The cells were pelleted, resuspended, and tested forviability by trypan blue exclusion, seeded and fed with 25 ml of theabove mentioned DMEM/Hams-F-12 supplemented medium. A factor VIIIrelated antigens assay, Grulnick et al., 1977, Ann. Int. Med.86:598-616, is used to positively identify endothelial cells, and silverstaining was used to identify tight junctional complexes, specific toonly small vessel endothelium.

[0102] Nylon filtration screen mesh (#3-210/36, Tetko, Inc., NY) wasprepared essentially as described above. The mesh was soaked in anacetic acid solution (1 ml glacial acetic acid plus 99 ml distilled H₂O)for thirty minutes, was rinsed with copious amounts of distilled waterand then autoclaved. Meshes were coated with 6 ml fetal bovine serum per8×8 cm mesh and incubated overnight. The meshes were then stacked, threehigh, and 3×10⁷ small vessel endothelial cells (cultured as described)were seeded onto the stack, and incubated for three hours at 37° C.under 5% CO₂ in a humidified atmosphere. The inoculated meshes were fedwith 10 ml of DMEM/Hams-F-12 medium every 3-4 days until completeconfluence was reached (in approximately two weeks).

[0103] 6.5 Establishment of the Three Dimensional Chondrocyte StromalCell Culture

[0104] Cartilage was harvested from articular surfaces of human joints.The cartilage pieces were digested with collagenase (0.2% w/v) incomplete medium (DMEM with 10% fetal bovine serum, glutamine,non-essential amino acids, sodium pyruvate, 50 μg/ml ascorbate and 35μg/ml gentamicin) for 20 hours at 37° C. Liberated chondrocytes werespun, resuspended in complete medium, counted and plated at 1×10⁶ cellsper T-150 flask. Cells were routinely passed at confluence (every 5-7days).

[0105] Polyglycolic acid mesh (1 mm diameter×2 mm thick) was sterilizedby ethylene oxide or electron beam treatment and presoaked overnight incomplete medium. The mesh was seeded in 6 well plates with 3-4×10⁶ cellsper mesh in a total volume of 10 μl and incubated for 3-4 hours at 37°C. in a tissue culture incubator. At this time, 1.5 ml of media wasadded. The seeded mesh was incubated overnight. 5 ml of media was addedthe next day. Media was changed three times per week until confluence isreached.

[0106] 7. Example: Extracellular Matrix Composition

[0107] The extracellular matrix has been characterized by a number ofanalytic methods to determine its content of matrix proteins, each valueis the average of at least two independent determinations. The matrixcontained type I and type III collagens, fibronectin, tenascin, sulfatedglycosaminoglycans, decorin and various other secreted humanextracellular matrix proteins. Additionally, the secreted matrixproteins were found throughout the three-dimensional support framework.The extracellular matrix contained a total protein amount of 292mg/cm²±0.06; fibronectin was present at 3.4 mg/cm²±1.2; and tenascin at−1.7 mg/cm²±0.6. Both fibronectin and tenascin showed the expectedmolecular weight distributions on immunoblots.

[0108] 7.1. Collagen Content of the Extracellular Matrix

[0109] Collagen content of the extracellular matrix was determined usingthe Sirius Red assay. The binding of Sirius Red F3BA in saturated picricacid solution has been used widely to estimate fibrotic collagendeposition. Bedossa et al., 1989, Digestion 44(1):7-13; Finkelstein etal., 1990, Br. J. Ophthalmol. 74(5):280-282; James et al., 1990, Liver10(1):1-5. The specificity of Sirius Red binding to collagen is basedlargely on its use as a histological stain. In rat liver with variousdegrees of cholestatic fibrosis, collagen content measured by Sirius Redbinding shows strong correlation with hydroxyproline content. Walsh etal., 1992, Analyt. Biochem. 203:187-190. In addition, histologicalstaining with Sirius Red is birefringent, indicating directional bindingrelated to the orientation of the collagen strands. Sirius Red is knownto bind to proteins other than the classical collagens that containcollagen-like triple helices, such as the complement component C1. Someminor binding to serum albumin has also been found, although controlexperiments using bovine serum albumin standard showed no interferencewith the assay. The interference is estimated to represent less than 2%of the collagen signal in the extracellular matrix and the use of SiriusRed assay gives a reproducible method for measuring collagens. Theextracellular matrix contained a collagen content of 0.61 mg/cm²±0.09.Further, collagens I and III showed the expected molecular weightdistributions on immunoblots.

[0110] 7.2. Collagen Fibers Visualized via Electron Microscopy

[0111] Collagen derived from the dermal tissue grown in vitro andcollagen derived from a normal adult human dermal sample were processedand visualized by transmission electron microscopy (TEM). Briefly, therespective collagens were weighed and placed in a sterile 50 mlcentrifuge tube with 30 ml 0.05 M Tris buffer, pH 8.0. After mixing fortwo hours on a wrist shaker, the Tris buffer was removed and thespecimen placed in a homogenization cylinder along with 30 ml fresh 0.05M Tris buffer. The sample was homogenized for 30 seconds in buffer aloneand then for two 30 second bursts following the addition of a dispersingagent as described in U.S. Pat. No. 4,969,912 and 5,332,802. Thetemperature was maintained at 5-10° C. during the mechanical disruptionprocess. The dispersing agent was added at a concentration of 0.05% (wetweight of the collagen). The homogenized preparation was centrifuged at3500 rpm for 6 minutes to separate the dispersed collagenous materialfrom the yet undispersed material. The undispersed residue was againtreated with dispersing agent at 0.05% (wet weight of the collagen) andhomogenized for two 30 second bursts. The dispersion was againcentrifuged to recover dispersed collagenous material which was added tothe first recovery.

[0112] The collagenous dispersion was filtered through a 100 micronfilter, centrifuged at 3500 rpm and the pellet was washed 3 times with0.004 M phosphate buffer, pH 7.4. The last centrifugation was conductedat 10,000 rpm to pack the collagenous pellet. Samples were thencollected for TEM. As shown in FIGS. 2A-D, the collagen fibers isolatedfrom either the extracellular matrix prepared from dermal tissue grownin vitro (FIGS. 2A-B) or from normal adult human dermis (FIGS. 2C-D)appeared identical in that intact collagen fibers with typical collagenbanding and normal periodicity in both preparations.

[0113] 7.3. Glycosaminoglycans Present in the Extracellular Matrix

[0114] Glycosaminoglycans have been shown to play a variety ofstructural and functional roles in the body and their presence in thesecreted extracellular matrix is important. Table II lists a number ofexamples of glycosaminoglycans which have been determined to be found inthe extracellular matrix as well as their functional importance innormal dermis. TABLE II NAME LOCATION GLYCAN FUNCTION MECHANISM VersicanMatrix 12-15 Structural Binds hyaluronic Chondroitin sulfate acid andcollagen Decorin Matrix 1 Chondroitin/dermatan Binding TGF-β Inactivatessulfate and other growth factors growth factors; binds to collagenBetaglycan Cell  1-4 TGF-β Type III Adjunct receptor membraneChondroitin/heparan receptor for TGFβ sulfate Syndecan Cell  1-3Chondroitin Growth factor membrane sulfate, 1-2 heparan binding sulfate

[0115] Further, the extracellular matrix was found to contain a total of2.8 mg/cm²±0.1 sulfated glycosaminoglycans.

[0116] 7.4. Growth Factors Present in the Extracellular Matrix

[0117] The cells producing and depositing the extracellular matrix alsoexpressed a number of different growth factors. Growth factors areimportant in the extracellular matrix for two reasons. During the growthof and deposition of the extracellular matrix, naturally seeded growthfactors help to control cell proliferation and activity. Further, growthfactors remain attached to the extracellular matrix. A variety of growthfactors have been determined to be expressed during the deposition ofthe matrix.

[0118] The expression of growth factors has been examined by polymerasechain reaction of reverse transcripts (RT-PCR) of total RNA. Briefly,RNA was extracted from the growing cells by an SDS precipitation andorganic solvent partition procedure. The RNA was transcribed usingsuperscript reverse transcriptase and random hexamer primers. The samebatch of reverse transcript was used for detection of all the growthfactors. PCR was performed under standard conditions, using 4 μl reversetranscript, corresponding to 200 ng RNA in a total volume of 20 μl.

[0119] Based on this assay, acidic and basic FGF, TGF-α and TGF-β, andKGF mRNA transcripts were present as were several others as shown inTable III, including PDGF, amphiregulin, HBEGF, IGF, SPARC and VEGF. Ofthese, PDGF and TGF-β3 are thought to be involved in regulation of cellproliferation and matrix deposition in culture, while TGF-β1, HBEGF,KGF, SPARC, VEGF and decorin are deposited in the matrix. Amphiregulin,IGF-1, IGF-2 and IL-1 were not expressed at the sensitivity used inthese experiments. TABLE III Messenger RNA Full Name Function ExpressionPDGF-A Chain Platelet- Mitogen for ++ derived growth fibroblasts,factor, A granulation chain tissue, chemotactic PDGF-B Chain Platelet-Mitogen for 0-(+)* derived growth fibroblasts, factor, B granulationchain tissue, chemotactic IGF-1 Insulin-like Mitogen for 0 growthfactor-1 fibroblasts IGF-2 Insulin-like Mitogen for (+) growth factor-2fibroblasts TGF-α Transforming Mitogen for + growth factor-αfibroblasts, keratinocytes Amphiregulin Amphiregulin Mitogen for 0fibroblasts, keratinocytes KGF Keratinocyte Mitogen for ++ growth factorkeratinocytes HBEGF Heparin- Mitogen for +++ binding fibroblasts,epidermal keratinocytes growth factor- like growth factor TGF-β1Transforming Stimulates + growth factor- matrix β1 deposition TGF-β3Transforming Stimulates ++—++ growth factor- matrix β3 deposition VEGFVascular Angiogenic ++ endothelial factor growth factor SPARC SecretedComplex anti- ++++ protein acidic angiogenic, and rich in angiogeniccysteine ICAM-1 Intercellular Lymphocyte + adhesion adhesion, molecule-1mobility VCAM Vascular Lymphocyte +++ cellular adhesion, adhesionmobility molecule GAPDh Glyceraldehyde Glycolytic +++ 3-phosphatehousekeeping dehydrogenase gene β2- β2- Antigen +++ microglobulinmicroglobulin presentation

[0120] The invention described and claimed herein is not to be limitedin scope by the specific embodiments herein disclosed since theseembodiments are intended as illustrations of several aspects of theinvention. Any equivalent embodiments are intended to be within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims.

[0121] A number of references are cited herein, the entire disclosuresof which are incorporated herein, in their entirety, by reference.

What is claimed is:
 1. A method for the production of a naturallysecreted extracellular matrix coated three-dimensional frameworkcomprising: (a) culturing cells on a three-dimensional framework underconditions favorable for cellular growth for a pre-determined timeperiod such that an extracellular matrix is secreted onto the inoculatedframework creating a coated framework; (b) killing the cells and (c)removing the cells and cellular debris.
 2. The method according to claim1 wherein the three-dimensional framework is made from a material, saidmaterial being selected from the group consisting of polyamide,polyester, polystyrene, polypropylene, polyacrylates, polyvinylcompounds, polycarbonate, polytetrafluorethylene, thermanox,nitrocellulose, cotton, polyglycolic acid, catgut suture material,cellulose, gelatin, chitosan, hyaluronic acid and dextran.
 3. The methodaccording to claim 1 wherein the three-dimensional framework has porespaces of about 150 μm to about 220 μm.
 4. The method according to claim1 wherein the extracellular matrix is secreted by tissue specific cells.5. The method according to claim 1 wherein the extracellular matrix issecreted by cells, said cells being selected from the group consistingof fibroblasts, osteoblasts, odontoblasts, chondrocytes, epithelialcells, smooth muscle cells, retinal cells, endothelial cells, stromalcells or combinations thereof.
 6. A method for the production of anaturally secreted extracellular matrix comprising (a) culturing cellson a three-dimensional framework under conditions favorable for cellulargrowth for a pre-determined time period such that an extracellularmatrix is secreted onto the inoculated framework creating a coatedframework; (b) killing the cells; (c) removing the cells and cellulardebris and (d) collecting the extracellular matrix deposited on thecoated framework.
 7. The method according to claim 6 wherein thethree-dimensional framework is made from a material, said material beingselected from the group consisting of polyamide, polyester, polystyrene,polypropylene, polyacrylates, polyvinyl compounds, polycarbonate,polytetrafluorethylene, thermanox, nitrocellulose, cotton, polyglycolicacid, catgut sutures, cellulose, gelatin, chitosan, hyaluronic acid anddextran.
 8. The method according to claim 6 wherein thethree-dimensional framework has pore spaces of about 150 μm to about 220μm.
 9. The method according to claim 6 wherein the naturally secretedextracellular matrix is secreted by tissue specific cells.
 10. Themethod according to claim 6 wherein the naturally secretedextracellular-iatrix is secreted by cells, said cells being selectedfrom the group consisting of fibroblasts, osteoblasts, odontoblasts,chondrocytes, epithelial cells, smooth muscle cells, retinal cells,endothelial cells, stromal cells or combinations thereof.
 11. Acomposition comprising a three-dimensional framework, said frameworkcoated with a naturally secreted extracellular matrix composed of humanproteins.
 12. The composition according to claim 11 wherein theframework is made from a material, said material being selected from thegroup consisting of polyamide, polyester, polystyrene, polypropylene,polyacrylates, polyvinyl compounds, polycarbonate,polytetrafluorethylene, thermanox, nitrocellulose, cotton, polyglycolicacid, catgut sutures, cellulose, gelatin, chitosan, hyaluronic acid anddextran.
 13. The composition according to claim 11 wherein thethree-dimensional framework has pore spaces of about 150 μm to about 220μm.
 14. The composition according to claim 11 wherein the naturallysecreted extracellular matrix is secreted by tissue specific cells. 15.The composition according to claim 11 wherein the naturally secretedextracellular matrix is secreted by cells, said cells being selectedfrom the group consisting of fibroblasts, osteoblasts, odontoblasts,chondrocytes, epithelial cells, smooth muscle cells, retinal cells,endothelial cells, stromal cells or combinations thereof.
 16. Aninjectable naturally secreted extracellular matrix composition for thetreatment of tissue defects comprising a human naturally secretedextracellular matrix and a pharmaceutically acceptable carrier.
 17. Thecomposition according to claim 16 wherein the naturally secretedextracellular matrix is produced by: (a) culturing cells on athree-dimensional framework under conditions favorable for cellulargrowth for a pre-determined time period such that an extracellularmatrix is secreted onto the inoculated framework creating a coatedframework; (b) killing the cells; (c) removing the cells and cellulardebris; (d) collecting the naturally secreted extracellular matrixdeposited on the coated framework and (e) processing the collectedextracellular matrix with a pharmaceutically acceptable carrier.
 18. Thecomposition according to claim 17 wherein naturally secretedextracellular matrices secreted by different tissue or cell types aremixed between steps (d) and (e), such that ratios of collagen types I-V,respective to each other, are adjusted.
 19. The composition according toclaim 16 wherein the framework is made from a material, said materialbeing selected from the group consisting of polyamide, polyester,polystyrene, polypropylene, polyacrylates, polyvinyl compounds,polycarbonate, polytetrafluorethylene, thermanox, nitrocellulose,cotton, polyglycolic acid, catgut sutures, cellulose, gelatin, chitosan,hyaluronic acid and dextran.
 20. The composition according to claim 16wherein the three-dimensional framework has pore spaces of about 150 μmto about 220 μm.
 21. The composition according to claim 16 wherein thenaturally secreted extracellular matrix is secreted by tissue specificcells.
 22. The composition according to claim 16 wherein the naturallysecreted extracellular matrix is secreted by cells, said cells beingselected from the group consisting of fibroblasts, osteoblasts,odontoblasts, chondrocytes, epithelial cells, smooth muscle cells,retinal cells, endothelial cells, stromal cells or combinations thereof.23. A method for the repair of tissue defects comprising injecting anaturally secreted extracellular matrix in a pharmaceutically acceptablecarrier at the site of the tissue defect.
 24. The method according toclaim 23 wherein the naturally secreted extracellular matrix is producedby: (a) culturing cells on a three-dimensional framework underconditions favorable for cellular growth for a pre-determined timeperiod such that an extracellular matrix is secreted onto the inoculatedframework creating a coated framework; (b) killing the cells; (c)removing the cells and cellular debris; (d) collecting the naturallysecreted extracellular matrix deposited on the coated framework and (e)processing the collected extracellular matrix with a pharmaceuticallyacceptable carrier.
 25. The method according to claim 23 whereinnaturally secreted extracellular matrices secreted by different tissueor cell types-are mixed between steps (d) and (e), such that ratios ofcollagen types I-V, respective to each other, are adjusted.
 26. Themethod according to claim 23 wherein the framework is made from amaterial, said material being selected from the group consisting ofpolyamide, polyester, polystyrene, polypropylene, polyacrylates,polyvinyl compounds, polycarbonate, polytetrafluorethylene, thermanbx,nitrocellulose, cotton, polyglycolic acid, catgut sutures, cellulose,gelatin, chitosan, hyaluronic acid and dextran.
 27. The method accordingto claim 23 wherein the three-dimensional framework has pore spaces ofabout 150 μm to about 220 μm.
 28. The method according to claim 23wherein the naturally secreted extracellular matrix is secreted bytissue specific cells.
 29. The method according to claim 23 wherein thenaturally secreted extracellular matrix is secreted by cells, said cellsbeing selected from the group consisting of fibroblasts, osteoblasts,odontoblasts, chondrocytes, epithelial cells, smooth muscle cells,retinal cells, endothelial cells, stromal cells or combinations thereof.